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目的评价健脾清化方对肾间质纤维化(RIF)的作用,初步探讨其作用的可能机制。方法成年SD大鼠54只,随机分为3组,即假手术对照组(C组)、UUO模型组(M组)和健脾清化方治疗组(T组),每组各18只。每组于术后第3、7和14天分批处死又分为N3、N7和N14三组,每组各6只。T组术后立即给予健脾清化方每日灌胃,C组、M组则分别予同体积生理盐水灌胃。术后第3、7和14天处死各组大鼠,分别取出左肾的1/2制作切片,行HE和Masson染色,观察肾脏病理学动态改变;免疫组化法检测肾间质组织内TGF-β1和Smad7、α-SMA蛋白的表达。半定量两步法RT-PCR测肾小管上皮细胞整合素连接激酶mRNA。结果 HE染色及Masson染色显示:M组术后随时间延长肾小管间质病变加重,程度同期最重,高于同期T组及C组;T组病变程度介于同期M组及C组之间;C组基本正常,结果有统计学意义(P<0.05)。免疫组化及RT-PCR结果:M组TGF-β1、α-SMA蛋白、ILK mRNA表达随时间延长逐渐上调,程度同期最高,高于同期T组及C组;T组蛋白表达程度介于M组及C组之间;C组表达最低,结果有统计学意义(P<0.05);M组肾间质Smad7的表达随时间延长逐渐下调,程度同期最低,低于同期T组及C组;同期T组肾间质Smad7表达明显上调,低于同期C组,但高于M组,结果有统计学意义(P<0.05);M组与T组各组内肾小管间质损害指数与TGF-β1、α-SMA蛋白及ILK mRNA表达呈正相关,Smad7蛋白表达与上述各指标呈负相关,结果有统计学意义(P<0.05)。结论健脾清化方对大鼠RIF有抑制作用,其机制可能通过增加大鼠UUO模型中Smad7蛋白的表达抑制TGF-β1/ILK信号转导,发挥抗大鼠RIF的作用。
Objective To evaluate the effect of Jianpi Qinghua Decoction on renal interstitial fibrosis (RIF) and to explore its possible mechanism. Methods Fifty-four adult SD rats were randomly divided into three groups: sham operation control group (C group), UUO model group (M group) and Jianpi Qinghua Decoction group (T group), with 18 rats in each group. Each group was sacrificed on days 3, 7 and 14 after operation and divided into three groups of N3, N7 and N14, 6 in each. T group immediately after giving Jianpi Qinghua Fang daily gavage, C group, M group were given the same volume of normal saline gavage. The rats were killed on the 3rd, 7th and 14th day after operation, and the sections of the left kidney were removed and made into sections. HE and Masson staining were performed to observe the pathological changes of the kidney. Immunohistochemistry was used to detect the expression of TGF -β1 and Smad7, α-SMA protein expression. Semi-quantitative two-step RT-PCR was used to detect integrin-binding kinase mRNA in renal tubular epithelial cells. Results The results of HE staining and Masson staining showed that the tubulointerstitial lesions of M group were more severe than those of T group and C group at the same period, and the severity of T group was between M group and C group ; C group was normal, the result was statistically significant (P <0.05). The results of immunohistochemistry and RT-PCR showed that the expression of TGF-β1, α-SMA protein and ILK mRNA in group M gradually increased over time, the highest in the same period and higher than that in the T and C groups; the protein expression in the T group was between M (P <0.05). The expression of Smad7 in M group decreased gradually with time prolonging, which was the lowest in the same period and lower than that in the T and C groups at the same period. In the same period, the expression of Smad7 in renal interstitium in T group was significantly higher than that in C group, but higher than that in M group (P <0.05); The tubulointerstitial damage index β1, α-SMA protein and ILK mRNA expression was positively correlated, Smad7 protein expression was negatively correlated with the above indicators, the results were statistically significant (P <0.05). Conclusion Jianpi Qinghua Decoction can inhibit the RIF in rats, and its mechanism may inhibit the signal transduction of TGF-β1 / ILK by increasing the expression of Smad7 in UUO rat model, which may play an anti-RIF role.